DNA polymerases are critically important enzymes and present targets for cancer chemotherapy that have not been adequately explored. Greater knowledge of the functions of individual eukaryotic DNA polymerases in DNA synthetic processes, and of their catalytic mechanisms, is significant for both fundamental biologic understanding and potential exploitation in cancer chemotherapeutic regimens. We propose here an investigation of eukaryotic DNA polymerases that takes advantage of our ability to create large libraries of mutant DNA polymerases with different catalytic properties. In Specific Aim 1, we will identify mutants of DNA polymerases-d, -e, and -a that incorporate specific, mutagenic nucleotide analogs. We will analyze the roles of these polymerases in DNA metabolism by replacing the endogenous polymerases with the corresponding mutant enzymes. Incorporation of the analog and/or induction of diagnostic mutations in cells undergoing replication, repair or recombination will provide a signature for the participation of each polymerase in these processes. In Specific Aim 2, we will utilize our new system for in vivo protein evolution to create novel DNA polymerase-6 mutants with enhanced capacity to copy DNA containing bulky lesions, and we will analyze the consequences of increased lesion bypass for cell survival and mutagenesis. In Specific Aim 3, we will analyze the mechanisms by which DNA polymerases select complementary nucleotide substrates by creating and studying mutator and anti-mutator mutants. The proposed studies should facilitate exploitation of specific DNA polymerases as chemotherapeutic targets by providing knowledge relevant to inhibiting their catalytic activities and/or disrupting their functions in cancer cells.